Trophectodermal cell-specific gene transfer methods

ABSTRACT

The present inventors discovered that genes could be introduced specifically into trophectodermal cells with high efficiency, by infecting blastocysts with viral vectors carrying an arbitrary polynucleotide, or by using a nucleic acid transfection reagent in blastocysts, from which zona pellucida (extracellular matrix covering preimplantation early embryos to protect them from infection of viruses and the like) is removed. This method has no risk of infecting cells of the inner cell mass, which develops into a fetus in the future, with the introduced polynucleotide because the trophectoderm serves as a barrier. The present invention provides methods for introducing foreign genes into only placenta but not fetus, which enables rescue of genetically mutant animals from embryonic lethality due to placental abnormality and allows their birth. Furthermore, it is possible to analyze expression and effect of genes that regulate placental formation or placental function by using these methods.

TECHNICAL FIELD

The present invention relates to methods for introducing arbitrary polynucleotides specifically into whole placenta. The present invention also relates to methods for producing non-human animals rescued from embryonic lethality. Furthermore, the present invention also relates to methods of screening for polynucleotides that rescue embryonic lethality.

BACKGROUND ART

Methods for producing transgenic mice using retroviral vectors (Non-Patent Document 1) are known as methods for introducing genes at the individual level using viral vectors. Transgenic mice can be efficiently produced by using retroviral vectors; however, a disadvantage is that silencing of gene expression occurs as a result of methylation of introduced genes, or the like (Non-Patent Document 2).

Long term expression of introduced genes can be expected with the lentivirus vector which is a retroviral vector, because it enables integration of foreign genes into chromosome. Furthermore, unlike typical retroviral vectors, lentivirus vectors have a nuclear translocation signal, and thus can introduce genes into non-dividing cells. When transgenic mice are produced using lentivirus vectors, the efficiency of production increases and gene silencing does not occur in long-term gene expression (Non-Patent Documents 3 to 5).

In these methods of transgenic mouse production, genes are introduced into most viral vector-infected fertilized eggs. Furthermore, these methods introduce genes into both placenta and embryo, and thus are regarded as very excellent techniques in developmental studies.

[Non-Patent Document 1] Proc. Natl. Acad. Sci. USA., 1985, September; 82(18): 6148-6152 [Non-Patent Document 2] Proc. Natl. Acad. Sci. USA., 1985, October; 82(20): 6927-6931 [Non-Patent Document 3] Mol. Cell. Biol., 2000, October; 20(20): 7419-7426

[Non-Patent Document 4] J. Virol., 2000, November; 74(22): 10778-10784

[Non-Patent Document 5] Proc. Natl. Acad. Sci. USA., 2002, Feb. 19; 99(4): 2140-2145

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

To analyze gene expression in placenta and its effects during embryogenesis, foreign genes must be introduced into only the placenta but not embryo. A conventional method for introducing genes into the placenta comprises direct injection of a viral vector or such into the placenta using an injection needle or the like after laparotomy during pregnancy. However, the disadvantages of the method are the great burden imposed by surgery and that the method cannot be expected to introduce genes into the entire area of placenta.

Furthermore, as LV-based methods for introducing genes into the placenta, a transgenic method using infection of fertilized eggs (two-cell stage embryos) and a method for microinjection into blastocyst cavity are known. The former method has an advantage that the operation is convenient and a disadvantage that genes are introduced into both placenta and embryo.

Meanwhile, although the latter method for microinjection into blastocyst cavity enables genes to be specifically introduced into the placenta, it is not generally used because the method requires an expensive system and skillful techniques. Furthermore, the method is problematic in that embryos are greatly damaged because a glass capillary is directly penetrated into blastocysts. In addition, it is difficult to treat embryos on a large scale by this method.

There is also a known method for preparing animals rescued from embryonic lethality due to placental abnormality, and it is based on a tetraploid rescue method. In this method, in addition to a mutant animal, two-cell stage embryos of a wild type animal are prepared and then fused together to prepare tetraploid embryos by electric treatment or such. Then, chimeric embryos are prepared by aggregating eight-cell stage embryos of the mutant animal with the tetraploid four-cell stage embryos of the wild type animal, from both of which the zona pellucida has been removed. Chimeric animals are created by transplanting the chimeric embryos into pseudopregnant animals.

However, experiments using this method are also time-consuming, because the method requires embryos of a wild type animal in addition to those of a mutant animal. In addition, the wild type embryos must be tetraploidized by electric fusion or such. Furthermore, the method is used for the purpose of complementing placental function in mutant animals and is thus not suitable for analyzing gene function in placenta.

The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide methods for introducing genes specifically into whole placenta.

Means for Solving the Problems

The present inventors conducted various examinations to achieve the objective described above. As a result, the inventors found that genes could be introduced specifically into trophectodermal cells with high efficiency by infecting blastocysts, from which zona pellucida (extracellular matrix covering preimplantation early embryos to protect them from viral infection and the like) has been removed, with viral vectors.

More specifically, the present inventors successfully introduced genes into the trophectoderm, which is the outer layer of blastocyst, by removing the zona pellucida with acid treatment, enzyme treatment, or physical treatment and co-culturing blastocysts with viral vectors. Thus, the inventors completed the present invention. Furthermore, the inventors found that this method has no risk of infecting cells of the inner cell mass, which develop into a fetus in the future, with the viral vector because the trophectoderm serves as a barrier. Specifically, the present invention provides the following [1] to [21]:

[1] a method for introducing an arbitrary polynucleotide specifically into a trophectodermal cell, which comprises the steps of:

-   (a) removing zona pellucida from a blastocyst; and -   (b) introducing an arbitrary polynucleotide into the blastocyst     obtained in step (a);

[2] the method of [1], wherein the arbitrary polynucleotide is a polynucleotide encoding a protein essential for fetal development or a polynucleotide that regulates expression of the protein; [3] the method of [2], wherein the polynucleotide encoding a protein essential for fetal development is at least a polynucleotide selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2;

[4] the method of any one of [1] to [3], wherein the blastocyst is derived from an animal having abnormality in a protein essential for fetal development or in the regulation of expression of the protein;

[5] a method for producing a blastocyst comprising a trophectodermal cell specifically introduced with an arbitrary polynucleotide, which comprises the steps of:

-   (a) removing zona pellucida from a blastocyst; and -   (b) introducing an arbitrary polynucleotide into the blastocyst     obtained in step (a);

[6] the method of [5], wherein the arbitrary polynucleotide is a polynucleotide encoding a protein essential for fetal development or a polynucleotide that regulates expression of the protein;

[7] the method of [6], wherein the polynucleotide encoding a protein essential for fetal development is at least a polynucleotide selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esxl, Arnt, Tcfeb, and Gjb2;

[8] the method of any one of [5] to [7], wherein the blastocyst is derived from an animal having abnormality in a protein essential for fetal development or in the regulation of expression of the protein;

[9] a method for producing a non-human animal, which comprises the steps of:

-   (a) removing zona pellucida from a blastocyst of a non-human animal; -   (b) introducing an arbitrary polynucleotide into the blastocyst     obtained in step (a); and -   (c) transplanting the blastocyst obtained in step (b) into a     recipient;

[10] the method of [9], wherein the arbitrary polynucleotide is a polynucleotide encoding a protein essential for fetal development or a polynucleotide that regulates expression of the protein;

[11] a method for producing a non-human animal, which comprises the steps of:

-   (a) removing zona pellucida from a blastocyst of a non-human animal     having abnormality in a protein essential for fetal development or     in the regulation of expression of the protein; -   (b) introducing a polynucleotide encoding the protein essential for     fetal development or a polynucleotide that normalizes the regulation     of expression of the protein into the blastocyst obtained in step     (a); and -   (c) transplanting the blastocyst obtained in step (b) into a     recipient;

[12] the method of [10] or [11], wherein the polynucleotide encoding a protein essential for fetal development is at least a polynucleotide selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Amt, Tcfeb, and Gjb2;

[13] a method for rescuing a non-human animal from embryonic lethality, which comprises the steps of:

-   (a) removing zona pellucida from a blastocyst of a non-human animal     having abnormality in a protein essential for fetal development or     in the regulation of expression of the protein; -   (b) introducing a polynucleotide encoding the protein essential for     fetal development or a polynucleotide that normalizes the regulation     of expression of the protein into the blastocyst obtained in step     (a); and -   (c) transplanting the blastocyst obtained in step (b) into a     recipient to give birth to a newborn animal;

[14] the method of [13], wherein the polynucleotide encoding a protein essential for fetal development is at least a polynucleotide selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esxl, Arnt, Tcfeb, and Gjb2;

[15] a method of screening for a polynucleotide that rescues embryonic lethality, which comprises the steps of:

-   (a) removing zona pellucida from a blastocyst of a non-human animal     having abnormality in a protein essential for fetal development or     in the regulation of expression of the protein; -   (b) introducing an arbitrary polynucleotide into the blastocyst     obtained in step (a); -   (c) transplanting the blastocyst obtained in step (b) into a     recipient to give birth to a newborn animal; and -   (d) selecting a polynucleotide that rescues embryonic lethality as     compared to when the arbitrary polynucleotide is not introduced;

[16] the method of any one of [9] to [15], wherein the non-human animal is selected from the group consisting of: mouse, rat, rabbit, dog, cat, bovine, horse, pig, goat, sheep, and monkey;

[17] the method of any one of [1] to [16], wherein the zona pellucida is removed by acid treatment, enzyme treatment, or physical treatment;

[18] the method of [17], wherein the acid treatment is a treatment with acidic Tyrode's solution;

[19] the method of [17], wherein the enzyme treatment is a pronase treatment;

[20] the method of any one of [1] to [19], wherein the polynucleotide is introduced by infecting a blastocyst with a viral vector carrying a desired polynucleotide; and

[21] the method of any one of [1] to [19], wherein the polynucleotide is introduced by using a nucleic acid transfection reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing structure of the LV vector (LV-CAG-EGFP) used in the present invention.

FIG. 2 shows photographs of fetuses and placentas of E13.5 embryos obtained by infection with an EGFP-expressing viral vector. The respective fetuses and placentas are: derived from non-treated embryo (top); obtained by TG method (middle); obtained by the method of the present invention (bottom). In the method of the present invention, expression of the introduced gene is found only in the placenta, while in the TG method, the gene expression is observed in both fetus and placenta.

FIG. 3 shows photographs of sections of placentas from E13.5 embryos obtained by infection with an EGFP-expressing viral vector. The respective thin sections of placenta are: derived from non-treated embryo (top); obtained by TG method (middle); obtained by the method of the present invention (bottom). In the placenta treated by TG method and the method of the present invention, gene expression was observed in almost all cells, except for maternal cells, in the giant cell layer, spongiotrophoblast layer, and labyrinth layer. a is a photograph after HE stain; b is a photograph under fluorescent microscope.

FIG. 4 shows diagrams and photographs illustrating rescue of erk2-knockout mice. a is a diagram illustrating the locus in erk2-knockout mice. The endogenous erk is not expressed, because a drug-resistance gene is inserted between exons 2 and 3. b is a diagram showing a lentivirus vector constructed to express erk2 under the control of a CAG promoter. c is a diagram showing genotypes of offsprings obtained by using the method of the present invention to transfer genes into blastocysts obtained by mating heterozygous mice. Homozygous mice are not born by natural mating; however, 16 homozygous mice were obtained by the placenta-specific gene transfer method of the present invention. d is a photograph showing PCR genotyping of the obtained offsprings. Homozygous mice were born, having only the mutant allele but no wild-type allele. e is a photograph showing erk2 wild-type mouse (left), heterozygous mouse (middle), and homozygous (right) newborn mouse born as a result of the treatment of the present invention. f is a photograph showing Western blot result. It was shown that the newborn expressed endogenous erk2 and homozygous mice did not express erk2.

FIG. 5 shows diagrams and photographs illustrating rescue of p38α-knockout mice. a is a diagram illustrating the locus in p38α-knockout mice. The endogenous p38α is not expressed, because a drug-resistance gene is inserted between exons 10 and 11. b is a diagram showing a lentivirus vector constructed to express p38α under the control of a CAG promoter. c is a photograph showing genotypes of offsprings obtained by using the method of the present invention to transfer genes into blastocysts obtained by mating heterozygous mice. Homozygous mice are not born by natural mating; however, 34 homozygous mice were obtained by the placenta-specific gene transfer method of the present invention. d is a photograph showing PCR genotyping of the obtained offsprings. Homozygous mice were born, having only the mutant allele but no wild-type allele. e is a photograph showing p38α wild-type mouse (left), and homozygous (right) newborn mouse born as a result of the treatment of the present invention. f is a photograph showing Western blot result. It was shown that the newborn expressed endogenous p38α, and homozygous mice did not express p38α.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is based on the present inventors' finding that arbitrary genes can be introduced specifically into trophectodermal cells with high efficiency, by infecting viral vectors carrying an arbitrary polynucleotide into blastocysts , from which zona pellucida (extracellular matrix covering preimplantation early embryos to protect them from viral infection and the like) has been removed, with viral vectors. More specifically, the present invention relates to methods for introducing arbitrary polynucleotides specifically into trophectodermal cells.

In the methods of the present invention, first, blastocysts are obtained. Blastocyst refers to an embryo that has finished the segmentation stage during early mammalian fetal development. Mammalian eggs are alecithal; they divide holoblastically and form aggregates of blastomeres. At the 32-cell stage, eggs are divided into trophectoderm which enfolds the outside of the aggregate and inner cell mass inside of the aggregate. The inner cell mass develops into the body of a fetus in the future, while the trophectoderm differentiates into the placenta. In the methods of the present invention, arbitrary polynucleotides are introduced specifically into trophectodermal cells that constitute the outmost layer of a blastocyst.

Blastocysts can be prepared by the methods described below. Specifically, eggs and sperms are collected from arbitrary animals, and then fertilization is carried out by methods known to those skilled in the art. Blastocysts can be prepared from the resulting fertilized eggs by methods known to those skilled in the art, for example, by culturing the eggs in kSOM medium for 96 hours. Alternatively, blastocysts can be obtained directly from animals according to methods that are conventionally used by those skilled in the art (for example, methods described in: Manipulating the mouse embryo, a laboratory manual, 3rd edition, p 201-203, Cold Spring Harbor Laboratory Press).

In the methods of the present invention, the zona pellucida is then removed from the blastocysts obtained above. Embryos (preimplantation early embryos) are covered with zona pellucida, which is extracellular matrix, to protect them from infection of viruses or the like. In the methods of the present invention, the zona pellucida is removed.

The zona pellucida can be removed by known methods, for example, acid treatment, enzyme treatment, or physical treatment. One example of such acid treatment is a treatment that uses acidic Tyrode's solution (Manipulating the mouse embryo, a laboratory manual, 3rd edition, p 485-486, Cold Spring Harbor Laboratory Press). The zona pellucida can be partially dissolved, for example, by sucking acidic Tyrode's solution (pH 2.3 to 2.5) with a micropipette and then gently spraying immobilized embryos with the solution. Alternatively, the zona pellucida can be dissolved by immersing embryos in acidic Tyrode's solution.

Enzyme treatment for removal of zona pellucida includes pronase treatment (Calbiochem 537088, Sigma P5147) and the like. For example, embryos are placed in a solution of 0.5% pronase until the zona pellucida dissolves. After the zona pellucida dissolves, the embryos are washed well with a medium, and then zona pellucida-removed blastocysts can be obtained (Manipulating the mouse embryo, a laboratory manual, 3rd edition, p 731, Cold Spring Harbor Laboratory Press).

Furthermore, in the present invention, zona pellucida can also be removed by physical methods. Such physical methods of removal include a zona pellucida dissection method in which embryos are immobilized and part of the zona pellucida is dissected with a micropipette (Hum. Reprod., 5:7-13, 1990), methods for dissecting, drilling, or thinning zona pellucida with a laser (Hum. Reprod., 15:1061-1064, 2000) or piezomicromanipulator (Developmental Biology, 250:348-357, 2002) and the like.

Zona pellucida can be removed by the methods described above. In the present invention, however, methods for removing zona pellucida are not limited to the examples described above, and the methods include all methods that can remove zona pellucida.

In the methods of the present invention, finally, arbitrary polynucleotides are introduced into zona pellucida-removed blastocysts. In a preferred embodiment, methods for introducing arbitrary polynucleotides into zona pellucida-removed blastocysts include methods for introducing a vector carrying an arbitrary polynucleotide into blastocysts, but are not particularly limited thereto.

Vectors carrying an arbitrary polynucleotide to be introduced into blastocysts include viral vectors. Such viral vectors include arbitrary viral vectors conventionally used by those skilled in the art, for example, lentivirus vectors (Molecular Therapy 2003, 8; 666-673), retroviral vectors (Molecular Therapy 2003, 8; 666-673), adenovirus vectors, HVJ liposomes, Lipofectamine, and the like. HVJ (hemagglutinating virus of Japan: Sendai virus) liposome is a vector which can efficiently introduce liposome-encapsulated genes and oligonucleotides to various organs in the body using activity of the fusion protein of HVJ, a virus causing cell fusion, and HMG-1, a DNA-binding protein.

Of such vectors, lentivirus vectors are preferred. Lentivirus belongs to the retrovirus family, and is an immunodeficiency virus in human, monkey, cat, and bovine. With respect to gene structure, the virus is constituted by several regulatory genes in addition to structural genes fundamental to the retrovirus (gag, pol, and env). Lentivirus vectors constructed by altering the lentivirus can integrate foreign genes into chromosome, and thus long term expression of the genes introduced therein can be expected. Furthermore, unlike other retroviral vectors, lentivirus vectors have a nuclear translocation signal, and thus can introduce genes into non-dividing cells.

Lentivirus vectors used in the present invention include lentivirus vectors having at least LTR, RRE, and GAG. Lentivirus vectors used in the methods of the present invention should have at least these requirements, but may additionally have other genes, for example, deltaU3, PPT, and WPRE. Such lentivirus vectors are also preferably used as the viral vector in the methods of the present invention.

In a particularly preferred embodiment, lentivirus vectors used in the present invention have LTR (deltaU3), GAG, RRE, PPT, and WPRE. A representative example of lentivirus vectors having such structures is a vector constructed by substituting a cDNA of interest for the GFP moiety of the GFP viral vector disclosed in Example 1 and the following document: Molecular Therapy 2003, 8; 666-673. The vector structure and construction method are disclosed in the document indicated above.

Blastocysts can be infected with a lentivirus vector carrying an arbitrary polynucleotide, for example, by mixing zona pellucida-removed blastocysts with a solution containing the lentivirus vector carrying the arbitrary polynucleotide, and then leaving the mixture to stand for 4 to 5 hours. By this method, a lentivirus vector having an arbitrary polynucleotide can be specifically introduced into the trophectoderm.

Alternatively, arbitrary polynucleotides may also be introduced into zona pellucida-removed blastocysts by using a nucleic acid transfection reagent. Herein, the nucleic acid transfection reagent refers to any reagent that can introduce an arbitrary polynucleotide into blastocysts. Such nucleic acid transfection reagents include, but are not limited to, for example, Lipofectoamine 2000 (Invitrogen), Effectene (Qiagen), and FuGene (Roche).

A polynucleotide to be introduced may be in a form such as DNA, RNA, cDNA, mRNA, or artificial nucleic acid. Such DNAs, RNAs, cDNAs, and mRNAs also include derivatives thereof. Such artificial nucleic acids include, but are not limited to, for example, DNAs, RNAs, cDNAs, mRNAs, in which their sugar chain structures are modified, or derivatives thereof. Furthermore, polynucleotides to be introduced may be naked polynucleotides or polynucleotides introduced into a vector. Those skilled in the art can design and use appropriate vectors depending on the purpose. Vectors used in the present invention may comprise, in addition to an arbitrary polynucleotide to be introduced, polynucleotide regions that function in expression hosts, such as transcriptional initiation site and transcription termination site, for more efficient expression of the arbitrary polynucleotide.

In a preferred embodiment of the present invention, polynucleotides to be introduced include, but are not particularly limited to, polynucleotides encoding a protein essential for fetal development or polynucleotides that regulate the expression of a protein essential for fetal development.

Such polynucleotides encoding a protein essential for fetal development include any polynucleotides that encode a protein directly or indirectly involved in the fetal development. Such polynucleotides include, but are not limited to, for example, Dlx3 (SEQ ID NO: 1), Fgfr2 (SEQ ID NO: 2), Fra1 (SEQ ID NO: 3), Fzd5 (SEQ ID NO: 4), Gab1 (SEQ ID NO: 5), Gcm1 (SEQ ID NO: 6), Grb2 hypomorph (SEQ ID NO: 7), Gja7 (SEQ ID NO: 8), Hgf (SEQ ID NO: 9), Hsp84-1 (SEQ ID NO: 10), Itgav (SEQ ID NO: 11), Junb (SEQ ID NO: 12), Lifr (SEQ ID NO: 13), ERK1 (SEQ ID NO: 14), ERK2 (SEQ ID NO: 15), ERK5 (SEQ ID NO: 16), MEK1 (polynucleotide encoding the amino acid sequence of SEQ ID NO: 17), MEKk3 (polynucleotide encoding the amino acid sequence of SEQ ID NO: 18), p38α (SEQ ID NO: 19), p38β (SEQ ID NO: 20), Met (SEQ ID NO: 21), Pdgfra (SEQ ID NO: 22), Pdgfb (SEQ ID NO: 23), Pparg (SEQ ID NO: 24), Rxra (SEQ ID NO: 25), Rxrb (SEQ ID NO: 26), Sos1 (SEQ ID NO: 27), Vhlh (SEQ ID NO: 28), Wnt2 (SEQ ID NO: 29), Ets2 (SEQ ID NO: 30), Mash2 (SEQ ID NO: 31), Egfr (SEQ ID NO: 32), Hsf1 (SEQ ID NO: 33), Bmp5 (SEQ ID NO: 34), Bmp7 (SEQ ID NO: 35), Dnmt1 (SEQ ID NO: 36), Itga4 (SEQ ID NO: 37), Lhx1 (SEQ ID NO: 38), Mrj (SEQ ID NO: 39), Tcf1 (SEQ ID NO: 40), Lef1 (SEQ ID NO: 41), Cdx2 (SEQ ID NO: 42), Eomes (SEQ ID NO: 43), Fgf4 (SEQ ID NO: 44), Esrrb (SEQ ID NO: 45), Hand1 (SEQ ID NO: 46), Mdfi (SEQ ID NO: 47), Esx1 (SEQ ID NO: 48), Arnt (SEQ ID NO: 49), Tcfeb (SEQ ID NO: 51), and Gjb (SEQ ID NO: 52).

Those skilled in the art can readily obtain the above-described polynucleotides encoding a protein essential for fetal development. For example, such polynucleotides can be isolated from natural sources, using various biological samples such as placental tissues, trophoblast stem cells, and differentiated cells thereof as a source, based on their physicochemical properties and the like. Alternatively, the polynucleotides may be chemically synthesized based on known sequence information. Alternatively, the polynucleotides can be obtained by using gene recombination techniques to transform host cells with a vector carrying polynucleotides encoding a protein essential for fetal development, then culturing the resulting transformed cells that produce the gene recombinant protein, and collecting the protein from the cells or culture supernatant thereof.

The above-mentioned polynucleotides encoding a protein essential for fetal development include homologous genes from various animals. Herein, “homologous gene” refers to the above-listed polynucleotides of SEQ ID NOs: 1 to 16 and 19 to 51, polynucleotides encoding a protein comprising the amino acid sequence of SEQ ID NO: 17 or 18, or polynucleotides encoding a protein having a biological function equivalent to that of the transcription/translation products of such polynucleotides, in various animals.

Methods that are well known to those skilled in the art for isolating homologous genes include hybridization techniques (Southern, E. M., Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, R. K., et al. Science, vol. 230, 1350-1354, 1985, Saiki, R. K. et al. Science, vol. 239, 487-491, 1988). More specifically, those skilled in the art can routinely isolate polynucleotides encoding a protein essential for fetal development from various animal cells and tissues (for example, placental tissues, trophoblast stem cells, and differentiated cells thereof), using as a probe, polynucleotides encoding a protein essential for fetal development (for example, the polynucleotide sequences of SEQ ID NOs: 1 to 16 and 19 to 51, and the polynucleotide sequences encoding a protein comprising the amino acid sequence of SEQ ID NO: 17 or 18) or a portion thereof, or using as a primer, oligonucleotides that specifically hybridize to the polynucleotides encoding a protein essential for fetal development. Alternatively, sequences of homologous genes may be obtained from known databases.

The polynucleotides of present invention encoding a protein essential for fetal development are preferably derived from, but are not particularly limited to, humans, mice, rats, rabbits, dogs, cats, bovines, horses, pigs, goats, sheep, and monkeys, more preferably from humans.

Furthermore, in a preferred embodiment, arbitrary polynucleotides used in the present invention include polynucleotides that regulate the expression of a protein essential for fetal development. Such polynucleotides include, for example:

-   (a) DNAs that expresses RNAs complementary to the transcription     product of a DNA encoding a protein essential for fetal development; -   (b) DNAs that expresses RNAs having a ribozyme activity of cleaving     specifically the transcription product of a DNA encoding a protein     essential for fetal development; -   (c) DNAs that expresses RNAs having an RNAi activity of cleaving     specifically the transcription product of a DNA encoding a protein     essential for fetal development; and -   (d) DNAs that bind specifically to a transcriptional regulatory     factor of a DNA encoding a protein essential for fetal development     and regulates expression thereof.

Herein, “regulation of expression of a protein essential for fetal development” includes enhancement or suppression of the transcription of a gene encoding a protein essential for fetal development, or enhancement or suppression of its translation into protein.

In an embodiment, “polynucleotide that regulates the expression of a protein essential for fetal development” is a DNA encoding an antisense RNA complementary to the transcription product of a DNA encoding a protein essential for fetal development. The antisense effect was for the first time proven using the transient gene expression method, based on the finding that antisense RNAs exert an antisense effect when introduced into plants by electroporation (Ecker and Davis, Proc. Natl. Acad. USA, 83: 5372, 1986). After this finding, other cases were reported, where the expression levels of target genes were reduced by antisense RNA expression in tobacco or petunia (Krol et al., Nature 333: 866, 1988). To date, the antisense effect has been established as a method for suppressing gene expression in eukaryotes, including both animals and plants.

The action of antisense nucleic acids in suppressing target gene expression includes: inhibition of transcription initiation by triplex formation; transcription suppression by hybrid formation with a site having a local open loop structure generated by RNA polymerase; transcription inhibition by hybrid formation with RNA as its synthesis advances; splicing suppression by hybrid formation at an intron-exon junction; splicing suppression by hybrid formation with the site of spliceosome formation; suppression of transport from the nucleus to the cytoplasm by hybrid formation with mRNA; splicing suppression by hybrid formation with the capping site or poly(A) addition site; suppression of translation initiation by hybrid formation with the translation initiation factor binding site; suppression of translation by hybrid formation with the ribosome binding site adjacent to the start codon; prevention of peptide chain elongation by hybrid formation with the translational region or the polysome binding site of mRNA; and suppression of gene expression by hybrid formation with the nucleic acid-protein interaction site. These inhibit the process of transcription, splicing, or translation and suppress target gene expression (Hirashima and Inoue, Shin Seikagaku Jikkenkoza 2 (New Lecture for Experimental Biochemistry 2), Kakusan IV (Nucleic Acid IV), Replication and Expression of Genes; Ed., Japanese Biochemical Society, Tokyo Kagaku Dojin Co., Ltd., pp. 319-347, 1993).

The expression of an endogenous protein essential for fetal development can also be suppressed by using ribozyme-encoding DNAs. The ribozyme refers to a RNA molecule with catalytic activity. There are various ribozymes with different activities, and particularly studies of ribozymes that serve as RNA-cleaving enzymes have enabled the design of ribozymes for site-specific RNA cleavage. Ribozymes include those comprising 400 nucleotides or more, such as group-I intron-type and M1 RNA included in RNAseP, as well as ribozymes with an active domain of about 40 nucleotides, called hammerhead- or hairpin-type ribozymes (Makoto Koizumi and Eiko Otsuka, Tanpakushitu, Kakusan, Koso (Protein, Nucleic acid and Enzyme), 35:2191, 1990).

For example, the autocleavage domain of hammerhead-type ribozymes cleaves G13U14C15 at the 3′ end of C15. The base pairing of U14 with A at position 9 is important for this activity, and cleavage has been shown to occur even when the nucleotide at position 15 is A or U, instead of C (Koizumi et al., FEBS Lett. 228:225, 1988). When the substrate-binding site of a ribozyme is designed to be complementary to an RNA sequence adjacent to the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU, or UA in the target RNA (Koizumi et al., FEBS Lett. 239:285, 1988; Makoto Koizumi and Eiko Otsuka, Tanpakushitu, Kakusan, Koso (Protein, Nucleic acid and Enzyme), 35:2191, 1990; Koizumi et al., Nucleic Acids Res. 17:7059, 1989).

Hairpin-type ribozymes are also useful for the purpose of the present invention. For example, hairpin-type ribozymes are found in the minus strand of satellite RNA in tobacco ringspot viruses (Buzayan, Nature 323:349, 1986). It has been demonstrated that this ribozyme can also be designed to cleave RNA in a target-specific manner (Kikuchi and Sasaki, Nucleic Acids Res. 19:6751, 1992; Yo Kikuchi, Kagaku To Seibutsu (Chemistry and Biology) 30:112, 1992).

In an alternative embodiment, “polynucleotide that regulates the expression of a protein essential for fetal development” is a DNA encoding double-stranded RNA (dsRNA) complementary to the transcription product of an endogenous DNA encoding a protein essential for fetal development. The introduction of a dsRNA comprising a sequence identical or similar to a target gene sequence into cells can cause the phenomenon called RNA interference (RNAi), where the expressions of both introduced foreign gene and target endogenous gene are suppressed. When about 40 to a few hundred base pairs of dsRNAs are introduced into cells, an RNase III-like nuclease called Dicer, which has a helicase domain, processes the dsRNAs from their 3′ end into about 21 to 23 base pairs of short interference RNAs (siRNAs) in the presence of ATP. Specific proteins bind to the siRNAs to form nuclease complexes (RNA-induced silencing complexes (RISC)). The complexes recognize and bind to the same sequence as siRNA, and then cleave the transcription product (mRNA) of a target gene at the position corresponding to the center of the siRNA by the RNaseIII-like enzymatic activity. In another pathway, the antisense strand of siRNA binds to mRNA and serves as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. Another pathway is also considered where this dsRNA also serves as a substrate for Dicer, producing new siRNAs to amplify the effect.

The RNAi was originally found in Caenorhabditis elegans (Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, 1998), and so far has been observed not only in Caenorhabditis elegans but also in various organisms such as plants, Nematoda, Drosophila, and Protozoa (Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., Caudy, A. A. & Hannon, G. J. Post-transcriptional gene silencing by double-stranded RNA. Nature Rev. Genet. 2, 110-119 (2001); Zamore, P. D. RNA interference: listening to the sound of silence. Nat Struct Biol. 8, 746-750 (2001)). Target gene expression has been confirmed to be suppressed when exogenous dsRNAs are introduced into these organisms. RNAi is also being used as a method for creating knockout animals

At the time when RNAi was disclosed, only a particular length (40 nucleotides) or longer of dsRNAs was believed to be effective. However, Tuschl et al. at the Rockefeller University in the U.S. reported that introducing about 21 base pairs of short-strand dsRNA (siRNA) into cells produces an RNAi effect without inducing PKR-mediated anti-viral response, not even in mammalian cells (Tuschl, Nature, 411, 494-498 (2001)). Since then, RNAi has drawn much more attention as a technology applicable to differentiated mammalian cells such as human cells.

A DNA of the present invention comprises an antisense-coding DNA which encodes an antisense RNA for any region within the transcription product (mRNA) of a target gene and a sense-coding DNA which encodes a sense RNA for any region within the same mRNA, and allows expression of the antisense RNA and the sense RNA from the antisense-coding DNA and the sense-coding DNA, respectively. Furthermore, a dsRNA can also be prepared from the antisense and sense RNAs. In the present invention, the target sequence is not particularly limited, as long as a dsRNA comprising a sequence identical or similar to the target sequence suppresses the expression of a DNA encoding a protein essential for fetal development when introduced into cells.

When an expression system for the dsRNA of the present invention is maintained in a vector or such, the construction may that that both antisense and sense RNAs are expressed from a single vector or independently from different vectors. For example, when both antisense and sense RNAs are expressed from a single vector, a construct can be prepared by separately constructing an expression cassette for the antisense RNA and an expression cassette for the sense RNA, where a promoter such as polIII series, which enables the expression of a short RNA, is linked upstream of the antisense-coding DNA and sense-coding DNA, respectively, and then inserting these cassettes into a vector in the same or reverse orientation.

Alternatively, the expression system may be constructed, such that the antisense-coding DNA and sense-coding DNA are arranged on separate strands and in the opposite direction. This construct comprises a double-stranded DNA (siRNA-coding DNA) in which the antisense RNA-coding strand and sense RNA-coding strand are paired with each other and promoters are arranged at both ends thereof in the opposite direction, such that the antisense RNA or sense RNA can be expressed from each strand. In this case, it is preferred that a terminator is arranged at the 3′ end of each strand (antisense RNA-coding strand or sense RNA-coding strand) to avoid addition of extra sequences downstream of the sense RNA and antisense RNA. A sequence of four or more consecutive adenine (A) nucleotides or the like may be used as the terminator. Furthermore, the two promoters are preferably different in this palindromic expression system.

Meanwhile, when the antisense RNA and sense RNA are expressed from separate vectors, constructs can be prepared, for example, by separately constructing an expression cassette for the antisense RNA and an expression cassette for the sense RNA, where a promoter such as polIII series, which enables the expression of a short RNA, is linked upstream of the antisense-coding DNA and sense-coding DNA, respectively, and then inserting these cassettes into separate vectors.

An siRNA may be used as the dsRNA for RNAi. “siRNA” refers to a double-stranded RNA consisted of short strands within a non-cytotoxic range, and is not limited to siRNAs with a full length of 21 to 23 base pairs as reported by Tuschl et al. (supra). The siRNA is not particularly limited, as long as its length is within a non-toxic range; for example, the length may be in the range of 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs. Alternatively, the final length of the formed double-stranded RNA portion of the expressed siRNA as a result of transcription may be, for example, in the range of 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs.

As the DNAs of present invention, constructs that form a double-stranded RNA with a hairpin structure (self-complementary “hairpin” RNA (hpRNA)) prepared by inserting an appropriate sequence (preferably intron sequence) between the inverted repeats of a target sequence (Smith, N. A., et al. Nature, 407: 319, 2000; Wesley, S. V. et al. Plant J. 27: 581, 2001; Piccin, A. et al. Nucleic Acids Res. 29:E55, 2001) can be used.

It is not necessary that DNAs used for RNAi be perfectly identical to the target gene; however, they have at least 70% or higher, preferably 80% or higher, and more preferably 90% or higher (for example, 95%, 96%, 97%, 98%, 99% or higher) sequence identity. The sequence identity can be determined by the methods described above.

Those skilled in the art can prepare the above DNAs of the present invention using common genetic engineering techniques. The DNAs of the present invention can be prepared, for example, by synthesizing arbitrary sequences using well known oligonucleotide synthesis methods.

The DNAs of the present invention can be introduced into cellular chromosomes and expressed within the cells as the way they are; however, the above DNAs are preferably carried by vectors for efficient gene transfer into cells, and the like. Vectors comprising the DNAs of the present invention are also included in the present invention.

Furthermore, in another embodiment, “polynucleotide that regulates the expression of a polynucleotide encoding a protein essential for fetal development” includes DNAs that regulate expression by binding specifically to a transcriptional regulatory factor for the DNA encoding a protein essential for fetal development. As an example of such DNAs, decoy DNAs are known. Decoy DNAs include, for example, about 20 nucleotides of DNAs comprising a sequence complementary to a transcriptional regulatory factor such as NF-κB. Decoy DNAs inhibit the function of a transcriptional regulatory factor by binding to the factor. As a result, the expression of a protein which is regulated by the transcriptional regulatory factor is also suppressed.

Thus, polynucleotides that are introduced specifically into the trophectoderm by the methods of the present invention include not only polynucleotides encoding a protein essential for fetal development but also polynucleotides that regulate the expression of a protein essential for fetal development. For example, a gene function in embryogenesis can be analyzed by introducing into blastocysts a polynucleotide that regulates the expression of a protein essential for fetal development.

It is possible to determine whether an arbitrary polynucleotide has been introduced specifically into the trophectoderm of a blastocyst by the methods of the present invention, by using methods known to those skilled in the art, for example, by amplifying introduced genes with PCR, or by detecting the expression of a reporter gene such as EGFP or lacZ with a fluorescence or luminescence method, or such.

Blastocysts to be introduced with the above polynucleotides encoding a protein essential for fetal development or polynucleotides that regulate the expression of a protein essential for fetal development are not particularly limited. Blastocysts to be introduced with such polynucleotides include, for example, blastocysts derived from animals that have abnormality in a protein essential for fetal development or in the protein expression regulation thereof. Herein, “protein essential for fetal development” refers to a protein directly or indirectly involved in fetal development, as described above. Specifically, the proteins include those comprising the amino acid sequences encoded by the nucleotide sequences of SEQ ID NOs: 1 to 16 and 19 to 51, and proteins comprising the amino acid sequence of SEQ ID NO: 17 or 18. Furthermore, “having abnormality in a protein essential for fetal development” refers a condition that the protein essential for fetal development is structurally or functionally abnormal. Alternatively, “having abnormality in the regulation of expression of a protein essential for fetal development” refers to having abnormality in either or both of the following processes: transcription of a DNA encoding a protein essential for fetal development into RNA and translation of said RNA into protein. Furthermore, in the present invention, “having abnormality in the regulation of expression of a protein” includes not only the case that production of the protein is completely inhibited due to the abnormality in either or both of the transcription and translation processes, but also the case where the level or timing of the protein expression is not normal or that the protein is produced with abnormality in part of the normal protein function or structure.

For example, animals that could not be naturally born due to embryonic lethality can be born by introducing a polynucleotide encoding a protein essential for fetal development into blastocysts of the animals having abnormality in the protein that is essential for fetal development or in the protein expression regulation thereof.

Animals from which blastocysts are derived include, but are not particularly limited to, humans, mice, rats, rabbits, dogs, cats, bovines, horses, pigs, goats, sheep, monkeys, and the like.

As described above, in the methods of the present invention, “polynucleotides that regulate the expression of a protein essential for fetal development” can also be introduced into blastocysts. Specific embodiments of such polynucleotides are described above. Furthermore, blastocysts into which such polynucleotides are to be introduced include blastocysts of animals that have abnormality in a protein essential for fetal development or in the protein expression regulation thereof, and blastocysts that do not have abnormality in essential proteins for fetal development or in the protein expression regulation thereof. When a polynucleotide that regulates the expression of a protein essential for fetal development is a polynucleotide that enhances the expression of a protein essential for fetal development, for example, animals that could not be naturally born due to embryonic lethality can be born by introducing the polynucleotide into blastocysts of animals having abnormality in a protein essential for fetal development or in the protein expression regulation thereof. Alternatively, when a polynucleotide that regulates the expression of a protein essential for fetal development is a polynucleotide that suppresses the expression of a protein essential for fetal development, for example, genes essential for fetal development can be identified, or gene function in fetal development can be analyzed by introducing the polynucleotide into blastocysts that do not have abnormality in essential proteins for fetal development or in the protein expression regulation thereof.

Thus, those skilled in the art can use the methods of the present invention for various purposes and applications by selecting appropriate polynucleotides to be introduced and appropriate blastocysts into which the polynucleotides are introduced. In the methods of the present invention, polynucleotides to be introduced and blastocysts into which the polynucleotides are introduced are not particularly limited. Furthermore, combinations of polynucleotide to be introduced and blastocyst into which the polynucleotide is introduced are not limited.

The present invention also relates to methods for producing blastocysts where an arbitrary polynucleotide is introduced specifically into the trophectodermal cells.

In the methods of the present invention for producing blastocysts, first, the zona pellucida is removed from blastocysts. Any blastocysts may be used. The blastocysts can also be obtained, for example, by the methods described above. Furthermore, the zona pellucida can be removed, for example, by the methods described above, but the methods are not limited thereto.

In the methods of the present invention for producing blastocysts, arbitrary polynucleotides are then introduced into the above blastocysts. The polynucleotides can be introduced by the methods described above, but the methods are not limited thereto. In the methods of the present invention for producing blastocysts, there is no limitation on the type of polynucleotide to be introduced, and any polynucleotides can be introduced. In a preferred embodiment, such polynucleotides include polynucleotides encoding a protein essential for fetal development or polynucleotides that regulate the expression of a protein essential for fetal development. Specific embodiments of these polynucleotides are also as described above.

Furthermore, there is no limitation on the type of blastocyst into which the above-described polynucleotides are introduced. For example, the above-described blastocysts may be used.

In the methods of the present invention for producing blastocysts, there is no limitation on the polynucleotide to be introduced, blastocyst into which the polynucleotide is introduced, or combinations thereof. Those skilled in the art can produce and use appropriate combinations depending on the purpose.

The present invention further relates to methods for producing non-human animals and methods for rescuing non-human animals from embryonic lethality.

In the methods of the present invention for producing non-human animals, first, the zona pellucida is removed from blastocysts of a non-human animal. Any blastocysts can be used for this purpose. The blastocysts to be used in the methods of the present invention for producing non-human animals are preferably, but are not limited to, blastocysts of non-human animals having abnormality in a protein essential for fetal development or in the protein expression regulation thereof. Polynucleotides can be introduced into such blastocysts to generate non-human animals rescued from embryonic lethality due to placental abnormalit.

Such blastocysts can be obtained by the methods described above. The zona pellucida may also be removed by any methods, for example, the methods described above.

In the methods of the present invention for producing non-human animals, an arbitrary polynucleotide is then introduced into the above blastocysts. The polynucleotide can also be introduced by the methods described above.

In the methods of the present invention for producing non-human animals, there is no limitation on the type of polynucleotide to be introduced. In a preferred embodiment, the polynucleotide includes polynucleotides encoding a protein essential for fetal development or polynucleotides that normalize regulation of the expression of a protein essential for fetal development. Specific embodiments of the polynucleotide encoding a protein essential for fetal development are as described above. Furthermore, the polynucleotides that normalize regulation of the expression of a protein essential for fetal development include polynucleotides that can correct abnormalities in either or both of the following processes: transcription of a DNA encoding a protein essential for fetal development to RNA and translation of the RNA to protein. Herein, normalization means not only perfect correction of the abnormality, but also partial correction of the abnormality and reduction in the degree of abnormality.

In the methods of the present invention for producing non-human animals, the above blastocysts are finally transplanted into a recipient. The recipient is preferably the same animal or an animal belonging to the same animal species as the animal from which the blastocysts are derived. Those skilled in the art can routinely transplant blastocysts into recipients (Manipulating the mouse embryo, a laboratory manual, 3rd edition, p 263-271, Cold Spring Harbor Laboratory Press). Non-human animals to be produced according to the present invention include, but are not limited to, for example, mice, rats, rabbits, dogs, cats, bovines, horses, pigs, goats, sheep, monkeys, and the like.

Thus, non-human animals which are embryonic lethal can be produced by using methods of the present invention for introducing of polynucleotides specifically into trophectodermal cells. It should be noted that the non-human animals produced by the methods of the present invention are not chimeric animals because they do not carry in their bodies the polynucleotide introduced into the blastocysts.

The present invention further provides methods for rescuing non-human animals from embryonic lethality. In the methods of the present invention for rescuing non-human animals from embryonic lethality, first, the zona pellucida is removed from blastocysts of a non-human animal having abnormality in a protein essential for fetal development or in the protein expression regulation thereof. Embodiments of non-human animals having abnormality in a protein essential for fetal development and non-human animals having abnormality in the regulation of expression of the protein essential for fetal development are as described above. Such blastocysts can be obtained, for example, by the methods described above. The zona pellucida may also be removed by any methods and the methods described above.

In the methods of the present invention for rescuing non-human animals from embryonic lethality, polynucleotides encoding a protein essential for fetal development or polynucleotides that normalize regulation of expression of a protein essential for fetal development are then introduced into the above blastocysts. The polynucleotides can also be introduced by the methods described above.

In the methods of the present invention for rescuing non-human animals from embryonic lethality, the above blastocysts are transplanted into a recipient to give birth to newborn animals. The recipient is preferably the same animal or an animal belonging to the same animal species as the animal from which the blastocysts are derived. Those skilled in the art can transplant blastocysts into recipients and give birth to newborn animals using conventional methods. Furthermore, non-human animals to be rescued by the methods of the present invention include, but are not limited to, for example, mice, rats, rabbits, dogs, cats, bovines, horses, pigs, goats, sheep, monkeys, and the like.

Furthermore, the present invention provides methods of screening for polynucleotides that rescue embryonic lethality, which comprise the following steps:

-   (a) removing the zona pellucida from blastocysts of a non-human     animal having abnormality in a protein essential for fetal     development or in the protein expression regulation thereof; -   (b) introducing arbitrary polynucleotides into the blastocysts     obtained in step (a); -   (c) transplanting the blastocysts obtained in step (b) into a     recipient to give birth to newborn animal; and -   (d) selecting polynucleotides that have rescued embryonic lethality     as compared to when no arbitrary polynucleotide has been introduced.

In the screening methods of the present invention, first, the zona pellucida is removed from blastocysts of a non-human animal having abnormality in a protein essential for fetal development or in the protein expression regulation thereof. Specific embodiments of non-human animals having abnormality in a protein essential for fetal development or in the protein expression regulation thereof are as described above. The removal from blastocysts can be achieved by the methods described above, but they are not limited thereto.

In the screening methods of the present invention, arbitrary polynucleotides are then introduced into blastocysts obtained as described above. Any polynucleotides may be introduced, and the length and origin thereof, and methods for obtaining them are not limited. Polynucleotides used in the screening methods of the present invention may be chemically synthesized, or derived from natural sources, for example, gene libraries such as cDNA libraries, and transcription products of the gene libraries. Furthermore, these polynucleotides can be used after appropriate labeling as needed. Such labeling includes, for example, radioactive labeling, fluorescent labeling, and the like. These polynucleotides can be introduced into blastocysts by the methods described above.

In the screening methods of the present invention, the above blastocysts introduced with an arbitrary polynucleotide are then transplanted into a recipient to give birth to newborn animals. As described above, blastocysts can be transplanted into recipients and newborn animals can be obtained by conventional methods used by those skilled in the art.

In the screening methods of the present invention, polynucleotides that have rescued embryonic lethality are selected as compared to when no arbitrary polynucleotide is introduced. The selected polynucleotides can serve as polynucleotides that normalize the regulation of expression of a protein essential for fetal development, and thus are useful in producing or rescuing non-human animals, which would naturally die due to embryonic lethality. Alternatively, these polynucleotides can be used as therapeutic agents for diseases caused by an abnormality in essential proteins for fetal development or in the expression regulation thereof.

All prior art documents cited herein are incorporated herein by reference.

EXAMPLES

Hereinbelow, the present invention will be described more specifically with reference to the Examples, but it is not limited thereto.

Materials and Methods (1) LV-Based Method of TG Production (Conventional Method)

PMSG (pregnant mare serum gonadotrophin) was administered to female BDF1 mice, and after about 48 hours, HCG was administered to induce superovulation. The mice were mated with male BDF1 mice. About 48 hours after mating, two- to four-cell stage embryos were washed out from the oviducts in FHM medium. The zona pellucida was removed by treatment with acidic Tyrode's solution. Then, the fertilized eggs were placed one by one in a spot of viral solution (1×10⁸ ng/ml) for infection. The eggs were cultured until they became blastocysts. After washing with kSOM medium, the blastocysts were transplanted into the uterus of a pseudopregnant mouse (on day 2.5 of pseudopregnancy).

(2) Tetraploid Rescue (Conventional Method)

Tetraploid refers to a tetraploid embryo. The embryo contributes mainly to the placenta which is an extraembryonic tissue. When an aggregation chimera is prepared with a tetraploid embryo and an embryo of a mutant mouse with placental abnormality, the mutant mouse can be developed in the placenta having normal functions.

PMSG was administered to female BDF1 mice, and after about 48 hours, HCG was administered to induce superovulation. The mice were mated with male BDF1 mice. About 48 hours after mating, two-cell stage embryos were washed out from the oviducts in FHM medium. Aggregation chimeras were produced by developing four-cell stage embryos from one tetraploid cell obtained by electrically fusing diploid two-cell stage embryos together; and placing them in close contact with eight-cell stage embryos obtained by superovulation treatment on the same schedule and mating of +/− heterozygotes, after zona pellucida was removed by treatment with acidic Tyrode's solution. The embryos were cultured in kSOM medium until they became blastocysts. The blastocysts were transplanted into the uterus of a pseudopregnant mouse (on day 2.5 of pseudopregnancy).

(3) Placenta-Specific Gene Transfer Method Using LV (Method of the Present Invention)

PMSG was administered to female +/− mice, and after about 48 hours, HCG was administered to induce superovulation. The mice were mated with male +/− mice. About 48 hours after mating, two- to four-cell stage embryos were washed out from the oviducts in FHM medium. The two- to four-cell stage embryos were cultured in kSOM medium for about 48 hours until they became blastocysts. After zona pellucida was removed by treatment with acidic Tyrode's solution, the blastocysts were placed one by one in a spot of viral solution (1×10³ ng/ml) for infection. The infection time was four to five hours. The blastocysts collected from the viral solution were washed once, and then the blastocysts were transplanted into the uterus of a pseudopregnant mouse (on day 2.5 of pseudopregnancy).

(4) Tissue Sections

Placentas were fixed with 4% paraformaldehyde at 4° C. for 12 hours, and then washed in PBS at 4° C. for two or three hours. PBS was replaced with acetone and after one or two hours, acetone was replaced with 1.5 ml/tissue of Technovit 8400 solution (0.06 g/10 ml). The tissues were fixed for about two hours. After the tissues precipitated, 50 μl of Technovit 8400 solution (small vial) was added. The tissues and resin were added inside the frame, covered, and then incubated at 4° C. for 12 hours for embedding. Thin sections were produced and observed.

(5) Western Blotting

Tails of the newborns were homogenized in lysis buffer using a homogenizer. The lysates were left on ice with occasional stirring for 1 hour, and then centrifuged at 13,000 rpm and 4° C. for 20 minutes. The resulting supernatants were used as extracts. The concentrations were determined, and adjusted to be constant. Then, the extracts were loaded and electrophoresed. After electrophoresis, the samples were transferred onto a PCDF membrane (Immobilon-P, Millipore, Bedford, Mass.). After blocking with TBS-T buffer containing 5% skim milk, the membrane was reacted with a primary antibody (ERK2, BD pan ERK #610123; p38α, Santa Cruz p38 (C-20) #sc-535) at 4° C. overnight. The membrane was washed three times with TBS-T buffer, and then incubated with an HRP-labeled anti-mouse antibody or anti-rabbit antibody at room temperature for one hour. After washing four times with TBS-T buffer, ECL™ (RPN2209) was used for color development and an image was developed.

(6) PCR

Genomic DNAs were extracted from a portion of the tissues of fetuses, placentas, and newborns to examine their genotypes by PCR method. Primers and PCR conditions used are shown below.

EGFP typing (SEQ ID NO: 52) Primer-1378: gagctagccaccatggtgagcaagggcgag (SEQ ID NO: 53) Primer-1382: tcaccttgatgccgttcttct PCR condition: 94° C. for two minutes, 30 cycles of (94° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds), and 72° C. for two minutes, followed by 4° C.

ERK2 typing (SEQ ID NO: 54) Primer-1331: taactgggtcgagcacagtgatgc (SEQ ID NO: 55) Primer-1332: tcagaattgatctggtcttcaagaccttg (SEQ ID NO: 56) Primer-1333: atgtatgctatacgaagttattagggc PCR condition: 95° C. for two minutes, 40 cycles of (95° C. for 20 seconds and 64° C. for 40 seconds), and 72° C. for one minute, followed by 4° C.

p38 typing (SEQ ID NO: 57) Primer-1380: ccctcgtggatggttgccagcagc (SEQ ID NO: 58) Primer-1343: cttactttgcactgaacacacacatcc PCR conditions: 94° C. for two minutes; 40 cycles of 94° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for one minute); and 72° C. for two minutes, followed by 4° C. The resulting PCR products were treated with the restriction enzyme EcoRV. Genotyping was based on the differences in size between the wild and mutant types.

Example 1 Infection and Evaluation of LV-CAG-EGFP

Introduction of a lentivirus (LV) carrying the GFP gene (LV-CAG-EGFP, which was constructed by the method described in Molecular Therapy 2003, 8;666-673; SIN lentivirus vector plasmid introduced with an EGFP-encoding cDNA under the control of a CAG promoter was constructed and combined with a packaging plasmid, Rev expression plasmid and VSVG expression plasmid; the mixture was introduced into 293T cells by the calcium phosphate method; viral particles released to the culture supernatant were collected and concentrated by ultracentrifugation; for the plasmids, see FIG. 1) was attempted using TG method and the method of the present invention. The gene used in this experiment was the GFP gene, and areas introduced with the gene emit green fluorescence. Subjects introduced with the gene by each method were examined for the infection site and evaluated for infection efficiency by microscopic observation, tissue sections, PCR, and such.

With the TG method, both E13.5 fetus and placenta emitted green fluorescence, and the gene was introduced into both (FIG. 2). Meanwhile, with the method of the present invention, the gene was introduced into only the placenta and not into the fetus (FIG. 2). A comparison of the sections in untreated placenta and placenta introduced with the gene by the method of the present invention demonstrated that the gene was introduced into the whole area of the placenta that had the gene introduction by the method of the present invention (FIG. 3). Furthermore, introduction of the gene was examined by PCR. With the TG method, the gene was introduced into only the placenta in some cases; however, in about 60% of all cases (38 of 66 subjects), the gene was introduced into both the placenta and fetus (Table 1). In contrast, when the same viral vector was introduced by the method of the present invention, the gene was introduced into only the placenta in all 69 subjects, and not into any fetus (Table 1). Furthermore, 91 newborns were analyzed, and the result showed that they were all born normally and the gene was not introduced (Table 1). The findings described above demonstrate that placenta-specific gene transfer can be achieved by the method of the present invention.

TABLE 1 Total Method dpc P + E P E (—) number TG 13.5 38 24 0 4 66 method 19.5 ND ND 28 25 53 Novel 13.5  0 69 0 0 69 method 19.5 ND ND 0 91 91 Screened by PCR However, E19.5 placenta was not analyzed. ND: no data P: Gene was introduced into only placenta E: Gene was introduced into only embryo (—): No gene was introduced

Example 2 Rescue of ERK2- and p38α-Knockout Mice

Next, the present inventors speculated that introducing a gene of interest by the methods of the present invention into mutant mice that were originally embryonic lethal due to placental abnormality could allow their development and postnatal analysis. ERK2-knockout mice and p38α-knockout mice were used for these experiments.

(1) ERK2-Knockout Mice (FIG. 4)

ERK2-knockout mice have been reported to be embryonic lethal around E10.5 due to placental abnormality (Hatano N. et al., Genes Cells. 2003 Nov; 8(11): 847-56). According to Hatano N. et al., labyrinthine layers are thin in the knockout placenta; the heart wall of the fetus becomes thinner and its development is retarded; and thus embryos after E11.5 are undetectable. The thinning of labyrinthine layers and heart wall abnormality were improved by the tetraploid rescue. Therefore, Hatano N. et al., argued that the heart wall thinning was a secondary abnormality as a result of the insufficient supply of oxygen and nutrient from the placenta.

A lentivirus vector was constructed to express ERK2 cDNA under the control of a CAG promoter (FIG. 3 b). After zona pellucida was removed from the blastocysts obtained by mating ERK2 +/− with ERK2 +/−, the blastocysts were infected with the viral vector and transplanted into the uterus of a pseudopregnant mouse. Genotyping of the resulting offsprings by PCR showed 16 homozygous mice (FIGS. 4 c and 4 d). The homozygous newborns seemed to be slightly small but healthy, and were also observed to drink milk (FIG. 4 e). Furthermore, Western blotting demonstrated that the ERK2 protein was absent in the mice (FIG. 4 f).

(2) p38α-knockout mice (FIG. 5)

p38α-knockout mice have been reported to be embryonic lethal around E10.5 due to placental abnormality (Adams R H et al., Mol Cell. 2000 July; 6(1): 109-16; Mudgett J S et al., Proc Natl Acad Sci USA. 2000 Sep. 12; 97(19): 10454-9). In p38α-knockout mice, placental labyrinthine layers and fetal heart wall are thin and imperfect, and aberrant cerebral angiogenesis is observed. When normal placenta was formed by tetraploid rescue in the mice, both of their heart wall and cerebral angiogenesis were normal. Adams R H et al. reported that non-placental abnormalities were assumed to be due to the insufficient supply of oxygen and nutrient from the placenta and thus the direct cause of embryonic lethality is placental abnormality.

In p38α-knockout mice, the gene was also introduced specifically into their placentas using a lentivirus vector (FIG. 5 b) and their genotypes were determined by PCR. As a result, 34 homozygous mice were born (FIGS. 5 c and 5 d). The homozygous deletion newborns seemed to be slightly small but healthy, and were also observed to drink milk (FIG. 5 e). Furthermore, Western blotting demonstrated that the p38α protein was absent in the mice (FIG. 5 f).

As described above, a system for placenta-specific gene transfer was established, and it was demonstrated that knockout mice which are embryonic lethal due to placental abnormality can be born by using this system. Furthermore, the use of knockout mice also enables postnatal functional analyses.

Forty or more types of genetically mutant mice exhibiting placental abnormality had been reported as of 2001 (Rossant J, Cross J C., Nat Rev Genet. 2001 July; 2(7):538-48. Review). When mice are embryonic lethal due to placental abnormality, the gene cannot be analyzed for its function or such after birth. The methods of the present invention can be applied to rescue experiments of such mutant mice and thus enable postnatal gene functional analyses.

Although tetraploid rescue merely compensates for the abnormal placenta of an embryo and normalizes its function, the use of LV allows mutant genes to be expressed in a placenta-specific manner, and LV can also be used in combination with RNAi. For example, it is expected that the placenta-specific function of ERK2 can be analyzed when a mutant ERK2 gene functions as dominant-negative mutant in the placenta.

Alternatively, it is also expected that by using LV, not only can each gene be analyzed in detail, but the relationship of the gene to other members of the gene subfamily and to genes upstream or downstream of the signaling pathway can also be examined. Thus, it is expected that with further studies, development of the LV-based placenta-specific gene transfer method can lead to applied research such as gene therapy and gene function analysis at the individual level.

INDUSTRIAL APPLICABILITY

The present invention provides methods for introducing genes specifically into whole placenta. The gene transfer methods of the present invention impose only a small burden on mothers and fetuses, because they do not introduce genes directly into placenta. Furthermore, unlike the microinjection system, the present methods require neither expensive devices nor skillful techniques.

In addition, the methods of the present invention can be used to introduce various genes into the placenta; this not only complements placental function in mutant animals, but also allows functional analyses of these genes in the placenta. Thus, the methods of the present invention are also useful for studies that analyze gene functions in embryogenesis. 

1.-21. (canceled)
 22. A method for introducing a polynucleotide specifically into a trophectodermal cell, which comprises the steps of: a) extracting a blastocyst from an animal; b) removing zona pellucida from the blastocyst; and c) introducing a polynucleotide into trophectodermal cells of the blastocyst by placing the blastocyst comprising trophectodermal cells obtained in step (b) in a solution comprising viral vectors; wherein the viral vectors introduce the polynucleotide only into the trophectodermal cells of the blastocyst.
 23. The method of claim 22, wherein the viral vector is selected from the group consisting of a retroviral vector and adenovirus vector.
 24. The method of claim 22, wherein the polynucleotide is a polynucleotide encoding a protein selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2.
 25. The method of claim 22, wherein the animal possesses abnormality in a protein essential for fetal development or in the regulation of expression of the protein.
 26. A method for introducing a polynucleotide specifically into a trophectodermal cell, which comprises the steps of: a) extracting a blastocyst from an animal; b) removing zona pellucida from the blastocyst; and c) introducing a polynucleotide into trophectodermal cells of the blastocyst with a nucleic acid transfection reagent; wherein the polynucleotide are introduced only into the trophectodermal cells of the blastocyst.
 27. The method of claim 26, wherein the polynucleotide is a polynucleotide encoding a protein selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2.
 28. The method of claim 26, wherein the animal possesses abnormality in a protein essential for fetal development or in the regulation of expression of the protein.
 29. A method for introducing a polynucleotide specifically into a trophectodermal cell, which comprises the steps of: a) extracting a blastocyst from an animal; b) removing zona pellucida from the blastocyst; and c) using a viral vector to introduce a polynucleotide only into trophectodermal cells of the blastocyte, wherein the polynucleotide encodes a protein selected from the group consisting of Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2; wherein the viral vectors introduce the polynucleotide only into the trophectodermal cells of the blastocyst. 